The leukocyte common antigen (CD45) family is a group of high molecular weight single transmembrane glycoproteins with cytoplasmic protein tyrosine phosphatase domains expressed on the plasma membranes of all leukocytes. Recent information regarding CD45 function indicates that antibody reagents to certain CD45 isoforms have potent immunosuppressive activities and may contribute to the immunosuppressive action of therapeutic agents such as anti-thymocyte globulin which is administered to transplant recipients for the prevention of graft-versus-host-disease and graft rejection. We have previously shown that certain extracellular domain epitopes of CD45 are involved in human neutrophil chemotaxis. We have pursued these observations using a genetic approach and mutagenized a murine macrophage cell line to obtain variant clones with low or undetectable CD45 expression. We found that cell line variants lacking CD45 expression were defective in their chemotactic response to recombinant human C5a, however, the cells exhibited normal random motility. These observations suggested that CD45 may be involved in directional (chemotactic) migration which is an essential host defense response to inflammatory stimuli. To clarify the role of CD45 in phagocyte chemotaxis, we are attempting to isolate a CD45 knockout mutant cell line. In a CD45 knockout mutant, reversion will be negligible and the function of CD45 can be evaluated by transfection with wild-type and mutant CD45 cDNA. During the year, lambda clones comprised of various fragments of the CD45 gene were screened with an exon 12 oligonucleotide probe and two positive clones were obtained. Detailed restriction maps were derived for each lambda clone and were used in the construction of a CD45 knockout vector with exon 12 disrupted by a neomycin gene. In an exon 12 knockout mutant, protein synthesis should terminate before the transmembrane region and result in a nonfunctional protein (gene). Cells have been transfected with the CD45 knockout vector and a PCR screening procedure for the detection of a mutant cell has been developed. Studies are underway to isolate the CD45 deficient mutant cell from the transfectants. The isolation of a CD45 deficient cell line will provide a valuable tool for establishing the functional role of the molecule in phagocyte chemotaxis and host defense.